Acetylation is one of the most crucial post-translational modifications affecting the protein function. Protein acetylation was catalyzed by acetyltransferase, and acetyl-CoA is the source of the acetyl group incorporated into certain lysine residues of proteins. Additionally, acetyl-CoA has been known to play critical roles to balance between carbohydrate metabolism and fatty acid synthesis. In this study, we have attempted to study whether lysine acetylation is an important process during adipocyte differentiation. Adipogenesis was significantly reduced by treatment with lysine deacetylase inhibitor TSA. Through acetylome analysis during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS analysis. Among these, malate dehydrogenase 1 (MDH1) is focused. The acetylation level of MDH1 increased up to 5-fold at late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induces the significant promotion of the adipogenesis. The introduction of mutations to putative lysine acetylation sites (especially at Lys-118) showed significantly loss of promotion effect of adipogenic differentiation. Furthermore, the acetylation of MDH1 induced the dramatic enhanced enzymatic activity and subsequently intracellular NADPH level. Additionally, the effects of acetylation of malate dehydrogenase 2 and prohibitin on adipogenesis were examined. The results clearly suggest that the adipogenic differentiation could be regulated by acetylation of various metabolic proteins and that the protein lysine acetylation is one of the cross-talk mechanisms between adipogenesis and intracellular physiological states.